Characterization of the receptor binding determinants of granulocyte colony stimulating factor

Young, Dennis C.; Cheng, Qi-Lin; Hou, Jinzhao; Matthews, David J.; Zhan, Hualin

doi:10.1002/pro.5560060823

Cited by 3 publications

(3 citation statements)

References 15 publications

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“…The far-ultraviolet (UV) circular dichroism (CD) spectra for met hG-CSF and the designed proteins were nearly identical to each other and to published spectra for met hG-CSF (Reidhaar-Olson et al 1996;Young et al 1997), indicating highly similar secondary structure and tertiary folds (data Fig. 1.…”

Section: Thermal Stabilitymentioning

confidence: 63%

“…Core residues were selected because optimization of these positions can improve stability yet minimize changes to the molecular surface, thus limiting the designed protein's potential for antigenicity. Ala scanning studies of G-CSF indicate one or two binding sites on the protein surface that are probably responsible for granulopoietic activity (Reidhaar-Olson et al 1996;Young et al 1997) (Fig. 1).…”

Section: Core Designsmentioning

confidence: 99%

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Development of a cytokine analog with enhanced stability using computational ultrahigh throughput screening

et al. 2002

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Granulocyte-colony stimulating factor (G-CSF) is used worldwide to prevent neutropenia caused by highdose chemotherapy. It has limited stability, strict formulation and storage requirements, and because of poor oral absorption must be administered by injection (typically daily). Thus, there is significant interest in developing analogs with improved pharmacological properties. We used our ultrahigh throughput computational screening method to improve the physicochemical characteristics of G-CSF. Improving these properties can make a molecule more robust, enhance its shelf life, or make it more amenable to alternate delivery systems and formulations. It can also affect clinically important features such as pharmacokinetics. Residues in the buried core were selected for optimization to minimize changes to the surface, thereby maintaining the active site and limiting the designed protein's potential for antigenicity. Using a structure that was homology modeled from bovine G-CSF, core designs of 25-34 residues were completed, corresponding to 10 21 -10 28 sequences screened. The optimal sequence from each design was selected for biophysical characterization and experimental testing; each had 10-14 mutations. The designed proteins showed enhanced thermal stabilities of up to 13°C, displayed five-to 10-fold improvements in shelf life, and were biologically active in cell proliferation assays and in a neutropenic mouse model. Pharmacokinetic studies in monkeys showed that subcutaneous injection of the designed analogs results in greater systemic exposure, probably attributable to improved absorption from the subcutaneous compartment. These results show that our computational method can be used to develop improved pharmaceuticals and illustrate its utility as a powerful protein design tool.

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Section: Thermal Stabilitymentioning

confidence: 63%

Section: Core Designsmentioning

confidence: 99%

Development of a cytokine analog with enhanced stability using computational ultrahigh throughput screening

et al. 2002

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“…New N-ends are distributed along the length of the hG-CSF coding sequence and are located not only between a helices but also within them. Since helix A and the protein segment between helix A and helix B are implicated in receptor contact [49][50][51], many new N-ends were purposely clustered in those regions.…”

Section: Cp Library Productionmentioning

confidence: 99%

Screening Libraries of Circularly Permuted Proteins by Phage Display to Manipulate Protein Topographies

Eteshola

Valkenburgh

Merlin

et al. 2005

Proceedings of the Institution of Mechanical Engineers, Part N:

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Active biomolecules, particularly proteins, are increasingly used in functional nanostructures. For some applications, native proteins (i.e. proteins as they occur in nature) are not effective nanodevice components because distances between the ends of the protein and some protein functional domain exceeds some critical nanoscale distance. This problem occurs in sensing architectures that detect energy or electron transfer where native protein sizes exceed critical distances for quantum energy transfer or exceed the thickness of shielding layers of counter ions, and might be addressed by alterations of protein topography. This work describes a high-throughput recombinant DNA method rapidly to identify functional circularly permuted (cp) protein variants. When appended to a sensing device or other nanometer scale distance-dependent device using chemoselective conjugation methods, cp variants offer a potential strategy to address the nanometer size limitations of native proteins.

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Site-Specific Thermodynamics: Understanding Cooperativity in Molecular Recognition

1998

Chem. Rev.

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Characterization of the receptor binding determinants of granulocyte colony stimulating factor

Cited by 3 publications

References 15 publications

Development of a cytokine analog with enhanced stability using computational ultrahigh throughput screening

Development of a cytokine analog with enhanced stability using computational ultrahigh throughput screening

Screening Libraries of Circularly Permuted Proteins by Phage Display to Manipulate Protein Topographies

Site-Specific Thermodynamics: Understanding Cooperativity in Molecular Recognition

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